Fluorescent molecules are of great interest because of their potential uses, for example, but not limited to, in labeling and detection of substrates or molecules in cell based assays, as components in organic electronic materials in molecular electronics, as pH sensors, and as metal sensors. There are currently several general classes of fluorescent molecules. These have been divided based on their structural motifs. For example, some common fluorescent structures include xanthene based fluorescein and rhodamine compounds, coumarins, pyrenes, and molecules based on the cyanine dyes. Other common fluorophores include, for example, auramine, acridine orange, dipyrrin, and porphyrin. The basic structures of these common fluorophores are presented in FIG. 1.
Molecular fluorescence is a type of photoilluminescence, which is a chemical phenomenon involving the emission of light from a molecule that has been promoted to an excited state by absorption of electromagnetic radiation. Specifically, fluorescence is a luminescence in which the molecular absorption of a photon triggers the emission of a second photon with a longer wavelength (lower energy) than the absorbed photon. The energy difference between the absorbed photon and the emitted photon results from an internal energy transition of the molecule where the initial excited state (resulting from the energy of the absorbed photon) transitions to a second, lower energy excited state, typically accompanied by dissipation of the energy difference in the form of heat and/or molecular vibration. As the molecule decays from the second excited state to the ground state, a photon of light is emitted from the compound. The emitted photon has an energy equal to the energy difference between the second excited state and the ground state.
Many fluorescent compounds absorb photons having a wavelength in the ultraviolet portion of the electromagnetic spectrum and emit light having a wavelength in the visible portion of the electromagnetic spectrum. However, the absorption characteristics of a fluorophore are dependent on the molecules absorbance curve and Stokes shift (difference in wavelength between the absorbed and emitted photon), and fluorophores may absorb in different portions of the electromagnetic spectrum.
The basic structures of the known fluorophores (FIG. 1) may be modified to provide different excitation and emission profiles. For example, two related compounds, fluorescein and rhodamine have different fluorescent characteristics. Fluorescein absorbs electromagnetic radiation having a wavelength of ˜494 nanometers (“nm”) and emits light having a wavelength at ˜525 nm, in the green region of the visible spectrum, whereas rhodamine B absorbs in radiation having a wavelength of ˜510 nm and emits light with an emission maximum of ˜570 nm, in the yellow-green region of the visible spectrum. Other fluorophores have different absorption and emission profiles. For example, coumarin-1 absorbs radiation at 360 nm and emits light at ˜460 nm (blue light); and pyrene absorbs radiation at ˜317 nm and emits light having a wavelength of ˜400 nm (violet light).
Exposure to lead is considered by the Center for Disease Control (CDC) as one of the United States' most serious environmental health threats for children. Exposure to lead may lead to damage to neurological, reproductive, and cardiovascular systems in children and adults and leads to other developmental problems. As a result of these concerns, the United States Environmental Protection Agency (EPA) has set a limit for lead in drinking water at 15 parts-per-billion (ppb). It is currently estimated that 1 in 11 children in the United States are at risk of adverse health effects from exposure to lead and that nearly 1.7 million children have lead blood levels of greater than 10 μg/dL.
Current methods for detecting and quantifying levels of lead in samples, such as environmental samples, include atomic absorption spectroscopy, inductively coupled plasma mass spectroscopy (“ICPMS”), and anodic stripping voltammetry which can measure lead at levels of ppb, ppb and parts-per-million (ppm), respectively. However, these methods have certain disadvantages, including being instrumentally intensive, generally expensive, lacking in spatial information, and restricted to in vitro measurements.
Despite their versatility, the known fluorophores have a number of disadvantages. For example, the absorption spectra of the fluoroscein class of fluorophores are generally pH sensitive, such that fluorescent yield decreases rapidly at pH levels below 8. Rhodamine and pyrene based fluorescent dyes are hydrophilic and hydrophobic, respectively.